Human sperm bind to the N-terminal domain of ZP2 in humanized zonae pellucidae in transgenic mice.

1Laboratory of Cellular and Developmental Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA..

Abstract

Fertilization requires taxon-specific gamete recognition, and human sperm do not bind to zonae pellucidae (ZP1-3) surrounding mouse eggs. Using transgenesis to replace endogenous mouse proteins with human homologues, gain-of-function sperm-binding assays were established to evaluate human gamete recognition. Human sperm bound only to zonae pellucidae containing human ZP2, either alone or coexpressed with other human zona proteins. Binding to the humanized matrix was a dominant effect that resulted in human sperm penetration of the zona pellucida and accumulation in the perivitelline space, where they were unable to fuse with mouse eggs. Using recombinant peptides, the site of gamete recognition was located to a defined domain in the N terminus of ZP2. These results provide experimental evidence for the role of ZP2 in mediating sperm binding to the zona pellucida and support a model in which human sperm-egg recognition is dependent on an N-terminal domain of ZP2, which is degraded after fertilization to provide a definitive block to polyspermy.

Human sperm bind to huZP2 rescue eggs. (A) Human sperm binding to human ZP1 (huZP1), huZP2, and huZP3 rescue and huZP4 transgenic eggs. Noninseminated human oocytes (human) and Zp3EGFP transgenic eggs (green zona) were positive and negative controls, respectively. Images obtained by confocal and DIC microscopy after staining with Hoechst were merged. Insets show magnification of a single humanized egg. (B) Same as A but with mouse sperm. Positive and negative controls were Zp3EGFP transgenic eggs and two-cell embryos, respectively. (C) 3D reconstruction of a z stack of confocal images of human sperm binding to the huZP2 rescue egg. Sperm heads binding to the zona pellucid were visualized by Hoechst staining, and the projection of sperm tails into the media was detected by single wavelength reflection. (D) Same as A but with human ZP1 (huZP1), huZP2, and huZP3 transgenic eggs before crossing into the corresponding null background. (E) Human sperm binding to huZP2 rescue eggs after sperm capacitation for 30 min, 2 h, 3 h, and 4 h. Confocal z projections (top) and DIC (bottom) images after staining with Hoechst.